Urotensin II (UII) is a peptide which acts on blood vessels. It produces vasodilative or vasoconstrictive effects depending on the vessel type and on the species concerned. In many respects, its activity is comparable with that of angiotension II (AII). Urotensin II, as well as its receptor GPR14, are expressed in the human coronary circulatory system, for example in endothelial cells, smooth muscle cells of coronary arteries, the myocardium or the coronary atheroma. Both factors are therefore ascribed an important role in cardiovascular organization at the cellular level and consequently in pathophysiological processes. Urotensin II is known to be the natural ligand of GPR14. GPR14 is a member of the GPCR (G protein-coupled receptors) group.
The use of urotensin II in experiments performed on anesthetized monkeys led to dramatic changes in the hemodynamic parameters. The increase in the peripheral resistance, together with a weakening of cardiac contractility and a reduced cardiac output, led to the coronary circulatory system collapsing. Comparable events also take place in humans.
G protein-coupled receptors (GPCRs) play a central role in a large number of very different physiological processes. It is assumed that about 1000 genes in the human genome encode this family of receptors. Roughly 40-50% of the presently available pharmaceuticals which can only be obtained on prescription act as agonists or antagonists of GPCRs. This underlines the important role of this class of receptors for industry carrying out pharmacological research. Because of the size and importance of this protein family, and in view of the fact that the physiological ligands for many GPCRs are not yet known (orphan GPCRs), it must be assumed that this class of receptors will in future be one of the most important reservoirs for suitable target proteins when searching for novel pharmaceuticals.
GPCRs constitute a family of integral membrane proteins which are located on the surfaces of cells. They receive signals from extracellular signaling substances (e.g. hormones, neurotransmitters, peptides and lipids) and transmit these signals, by way of a family of guanine nucleotide-binding proteins, what are termed the G proteins, into the interior of the cell. In doing so, they activate a variety of signal transduction pathways depending on the specificity of the receptor, on the G protein which is activated and on the cell type.
The polypeptide chains of all GPCRs fold into seven ∀ helices, which span the phospholipid double layer of the cell membrane. The seven transitions of the membrane give rise to extracellular and intracellular loops which make it possible to bind ligands extracellularly and to couple on G proteins intracellularly. For this reason, GPCRs are also termed seven-transmembrane receptors.
All G protein-coupled receptors function in accordance with a common basic pattern: the binding of an extracellular ligand leads to a change in the conformation of the receptor protein, thereby enabling the latter to make contact with a G protein. G Protein-mediated signal transduction cascades within the cell lead ultimately to the cell making a biological response.
G proteins are heterotrimeric proteins which are composed of the subunits ∀, ∃ and (and, due to lipid anchors, are located on the inner side of the cell membrane. The coupling of activated GPCRs to G proteins brings about a GDP/GTP exchange on the G∀ subunit and the dissociation of the heterotrimer into an ∀ subunit and a ∃γ subunit. Both the activated ∀ subunit and the ∃γ complex are able to influence intracellular effector proteins.
Activation of the membrane-located adenylate cyclase (AC) by G proteins of the G∀s type leads, for example, to an increase in the intracellular level of cAMP, while activation by G proteins of the G∀i type leads to a fall in this level. G proteins of the Gq type activate phospholipase C (PLC), which catalyzes the formation of inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG). These molecules in turn lead to Ca2+ being released from intracellular stores or to protein kinase C (PKC) being activated, with further effects in both cases. In addition to the abovementioned G protein types (G∀i/s, Gq), still further types exist, with these types being designated G16, G12/13, etc. The multiplicity of these G proteins is reflected in the large number of very different functions possessed by GPCRs.
The mouse urotensin II receptor is disclosed in WO 00/75168. Methods for identifying agonists and antagonists for GPR14 are likewise disclosed, for example, in WO 99/40192.
The prior art with regard to screening methods has so far not yet taken any account of the fact that the urotensin II receptor can be used specifically for finding those compounds which are genes or gene products which are involved in the synthesis, structure or breakdown of the extracellular matrix. As a result, it is not possible to search effectively enough, from the pharmaceutical point of view, for substances which counteract diseases which are accompanied by an increase in connective tissue.
The citation of any reference herein should not be construed as an admission that such reference is available as “Prior Art” to the instant application.